Malaria remains one of the most devastating parasitic diseases in the world with the vast majority of deaths caused by Plasmodium falciparum. The pathology of the disease results exclusively from the blood-stage of the infection during which parasites invade and multiply within host erythrocytes. To establish this intracellular niche, P. falciparum imposes striking modifications to the permeability, rigidity and cytoadherent properties of the host cell. This remodeling is accomplished through the export of numerous effector proteins into the cytosol and membrane of the erythrocyte. During a final step in the export process, effector proteins must cross a vacuole membrane surrounding the parasite to enter the host cell but the mechanism for this remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been identified and suggested to fulfill this role; however, direct functional evidence is lacking. To explore PTEX function, we have generated a conditional mutant of heat shock protein 101 (PfHSP101), an AAA+ ATPase component of the translocon, by fusion of a destabilization domain to the endogenous PfHSP101 C-terminus. In the absence of a stabilizing ligand, this strain experiences a complete block in protein export and growth. Related AAA+ proteins can unfold and directionally translocate substrates through a central channel, suggesting PfHSP101 may drive recognition of effector proteins and power the export process. This proposal aims to determine the role of PfHSP101 in effector export by examining the biochemical and functional properties of PfHSP101 in vitro and in live parasites. Specific Aim 1 seeks to understand the catalytic and substrate recognition properties of recombinant PfHSP101 and to dissect the role of PfHSP101 in protein export within intact parasites using the conditional mutant. Specific Aim 2 will exploit the conditional mutant to determine the first exported proteome, enabling identification of novel effectors. Preliminary results highlight PfHSP101/PTEX as a novel drug target and the proposed experiments will reveal key mechanistic information about the role of PfHSP101 in export as a necessary basis for rational drug design. Furthermore, forward identification of novel exported proteins with roles in parasite survival and virulence may provide needed additional therapeutic targets. Collectively, the results of this work will support development of new tools for control of malaria disease.